RESUMO
In this Research Communication we evaluate the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify 380 bacteria isolated from cases of bovine mastitis in Brazil. MALDI-TOF MS identifications were compared to previous identifications by biochemical tests and 16S rRNA sequencing. MALDI-TOF MS achieved a typeability of 95.5%. The accuracy of MALDI-TOF MS for the identification of Staphylococcus isolates was 93.2%. The agreement between MALDI-TOF MS and biochemical identification of Streptococcus agalactiae was 96%, however, the agreement between these techniques for identifying other catalase-negative, Gram-positive cocci was lower. Agreement in identifying Gram-negative bacteria at the genus level was 90.5%. Our findings corroborate that MALDI-TOF MS is an accurate, rapid and simple technique for identifying bovine mastitis pathogens. The availability of this methodology in some research institutions would represent a significant step toward increasing the diagnosis and epidemiological studies of bovine mastitis and other animal infectious diseases in Brazil.
Assuntos
Mastite Bovina/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Animais , Brasil , Bovinos , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Leite/microbiologia , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus/genética , Streptococcus agalactiae/genéticaRESUMO
We compared the potential of routine techniques used for the identification of Staphylococcus species, aiming to evaluate their accuracy in the detection of 43 Staphylococcus chromogenes strains isolated from bovine mastitis that, despite being a coagulase-negative species, are able to clot plasma. These strains could be mistakenly suspected to be S. aureus and lead to an unappropriated treatment of the disease. MALDI-TOF, PCR-RFLP of the chaperonine gene groEL, and sequencing of the 16S rRNA and elongation factor Tu gene tuf were employed. Results from the four methods were coincident for only half of the strains because of the low accuracy of the groEL PCR-RFLP (51.2% accuracy). Even though all the sequencing results were identical, the high accuracy of the MALDI-TOF results (97.7% accuracy, with only one strain misidentified) encourage the use of this technique, since it does not require laborious sample preparation, being fast and simple to perform.(AU)
Nós comparamos o potencial de técnicas rotineiras utilizadas para a identificação de espécies de Staphylococcus, com o objetivo de avaliar a acurácia delas na detecção de 43 isolados de Staphylococcus chromogenes envolvidos com mastite bovina que, apesar de ser uma espécie coagulase-negativa, são capazes de coagular o plasma. Essas cepas poderiam ser erroneamente suspeitas de serem S. aureus e levarem a um tratamento não adequado da doença. MALDI-TOF, PCR-RFLP do gene da chaperonina groEL e sequenciamento do gene do rRNA 16S e do gene do fator de elongação Tu, tuf, foram avaliados. Os resultados dos quatro métodos foram coincidentes para apenas metade das cepas, devido à baixa precisão da PCR-RFLP com groEL (51,2% de acurácia). Apesar de todos os resultados do sequenciamento serem idênticos, a alta precisão dos resultados do MALDI-TOF (97,7% de acurácia, com apenas uma cepa identificada incorretamente) encoraja o uso dessa técnica, pois, não requer preparação laboriosa de amostras, sendo rápida e simples de executar.(AU)
Assuntos
Animais , Bovinos , Staphylococcus , Mastite Bovina/diagnóstico , Mastite Bovina/sangue , Testes de Coagulação SanguíneaRESUMO
To characterize 73 Staphylococcus aureus isolates from infections in an orthopedic hospital in Rio de Janeiro, we investigated the SCCmec types, the clonality by pulsed field gel electrophoresis and multilocus sequence typing, and the presence of virulence genes. Twenty-eight (38.3%) methicillin-resistant (16 SCCmec type IV and 12 type III) isolates were detected. Most (83.5%) of the isolates were included in five lineages: sequence type (ST) 239 (SCCmecIII), 1, 5, 30, and 1462 (SCCmecIV and/or methicillin-susceptible isolates). Virulence genes fnbB, bbp, and pvl were related to STs 239, 30, and 30/1462, respectively. Isolates from STs 1, 5, and 30 presented specific virulence profiles, irrespective of methicillin resistance.
Assuntos
Genes Bacterianos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Fatores de Virulência/genética , Eletroforese em Gel de Campo Pulsado , Humanos , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Tipagem de Sequências Multilocus , Infecções Estafilocócicas/microbiologia , VirulênciaRESUMO
Change in epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) was observed because of the emergence of infections by non-multiresistant MRSA (nMRSA) in our hospital in Rio de Janeiro, Brazil. Clinical characterization and molecular analysis of 20 nMRSA isolates recovered from 17 patients, between February 2005 and March 2006, were performed. The analysis included SCCmec (staphylococcal cassette chromosome mec), pulsed field gel electrophoresis (PFGE), multilocus restriction fragment, and multilocus sequence typing. MICs for oxacillin and vancomycin and presence of Panton-Valentine leukocidin (PVL) genes were also investigated. All but 1 of the 20 isolates presented SCCmec type IV. PFGE clustered all isolates into 9 genotypes. MIC < or = 16 microg/mL to oxacillin was found for 65% of the isolates, whereas 80% exhibited MIC of 2 microg/mL for vancomycin. PVL-encoding genes were observed in 3 isolates. Polyclonal presence of nMRSA SCCmec IV was observed in our institution, including community and health care-associated isolates, which belonged to the sequence types (STs) 1 (clonal complex [CC1]), ST5 (CC5), ST8 and ST72 (CC8), ST97 (CC97), and 2 ST singletons (SLV5 and SLV30).
Assuntos
Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Adolescente , Adulto , Idoso , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Técnicas de Tipagem Bacteriana/métodos , Brasil/epidemiologia , Análise por Conglomerados , Impressões Digitais de DNA/métodos , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado/métodos , Exotoxinas/genética , Feminino , Genótipo , Hospitais , Humanos , Leucocidinas/genética , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Adulto JovemRESUMO
Molecular aspects of heat-shock response were investigated in monogenetic and digenetic members of the Trypanosomatidae and the data obtained compared. Trypanosoma cruzi and Crithidia fasciculata differ in the number of heat-shock proteins (HSPs) induced and in the range of supra-optimal temperature induction of these proteins. Whereas low molecular weight Hsps were induced by high temperature in Crithidia, this effect was only seen in T. cruzi after ethanol treatment. The 61 kDa peptide of T. cruzi, induced by heat, was characterized as a HSP60 family member by Western blot using a Mycobacterium polyclonal anti-HSP60 antibody. The HSP61 aa. sequence, deduced from the isolated HSP60 gene and its mRNA product were characterized. The predicted aa. sequence has shown the presence of a mitochondrial peptide leader and no large domains of aa. sequence conservation were found when compared to other known HSP60, in contrast to what is observed in HSP70. Furthermore, the HSP60 gene is apparently conserved in T. cruzi, C. fascilulata and Leishmania as suggested by genomic Southern blot analysis.